1. Field of the Invention
The present invention, in the fields of virology and molecular biology, relates to nucleic acid, methods and polypeptides relating to target cell specific delivery of therapeutic and/or diagnostic agents by target cell specific delivery vectors. Target cells are made specific for such delivery vectors by their (i) association with a chimeric viral receptor polypeptide having a second virus host cell range different from the target cell viral host cell range and/or (iii) providing a delivery vector having a binding domain specific for the target cell.
2. Description of the Background Art
Cell specific delivery of therapeutic or diagnostic agents in animals and human patients has suffered from the problems of non-tissue specific delivery, detrimental side effects and lack of effective dosage delivered to the target cells. While the use of monoclonal antibodies as diagnostic agents, therapeutic agents or delivery vectors overcomes the problem of tissue specificity, monoclonal antibodies suffer from the problem of immunogenicity, as well as insufficient dosage, due to clearance of antibody and/or fragments thereof by the kidneys or liver.
As an alternative delivery vector, the use of recombinant viruses has been investigated to determine suitable delivery vectors for therapeutic or diagnostic agents.
Viruses infect a cell by first binding their viral binding domain to a cell at the cell's viral receptor. The viral binding domain of the virus binds the cell's viral receptor at the viral binding site. A number of virus-specific cellular receptors have been identified, and most of these viral receptors have other known cellular functions for the cell. The degree of expression of such viral binding receptors in cells is thus a strong determinant of susceptibility of these cells to viral infection. Binding to the viral receptor is required for fusion of the virus envelope protein (env) to the target cell at the cell surface. (White et al., Quant. Rev. Biophys. 16: 151-195 (1983)). After fusion of the viral binding domain via viral binding to the cell's viral receptor binding site, the viron core enters the cytoplasm of the bound cell and the viral replication process is initiated. In some cases viruses bound to their receptors can also enter cells by receptor mediated endocytosis, such as by some retroviruses.
Retroviruses may be classified by the range of species that they are able to infect. For example, the mouse type C retroviruses, which are used in genetic engineering, fall into five general classes: amphotropic, which exhibit the broadest host range, encompassing a diversity of mammalian species including humans and rodents: polytropic, which use a receptor distinct from that for amphotropic viruses and also exhibit a more restricted host range; 10A1, which use both the amphotropic receptor as well as a second, widely expressed, receptor (Ott, et al., J. Virol (1990), J. Virol 19:13-18 (1976)); xenotropic, which also have a wide mammalian species host range but cannot infect mouse cells; ecotropic, which infect only rodent species or any combination thereof. Of these five mouse retrovirus receptor classes, only the receptor for the ecotropic Moloney murine leukemia virus (MLV) (see, e.g., Albritton, et al., Cell 57:659-666 (1989) has been cloned.
Human cells have been characterized at having at least eight distinct receptors for retroviruses (Sommerfelt, et al., Virology 176:58-69 (1990)). Of these, at least two now been cloned: the receptor for human immunodeficiency virus, a lentivirus (Maddon, Cell 47:333-348 (1986), McDougal, et al., Science 231:382-285 (1986), and the receptor for gibbon ape leukemia virus, a type C Retrovirus (O'Hara, et al. Cell Growth Differ. 1:119-127 (1990). The receptor used by human immunodeficiency virus, the CD4 antigen, is similar to receptors used by a number of other viruses in that it is a member of the immunoglobulin superfamily of cell surface proteins. In contrast, the gibbon ape leukemia virus receptor is not an immunoglobulin-like protein (O'Hara, et al., Cell Growth Differ. 1:119-127 (1990)).
Although amphotropic mouse retroviruses have a broad tropism, there are restrictions to their host cell range, both phylogenetic and based on tissue distribution. While many human cell types are infectable by amphotropic murine retroviruses, cell lines derived from certain mammalian species tissues, (e.g., bovine kidney cell line MDBK and Chinese hamster ovary cell line CHO-K1), are not infectible et al., J. Virol. 19:19-25 (1976), Rasheed, et al., J. Virol. 19:13-18 (1976)). Other tissues, such as, but not limited to, human lymphoid cells, are also poorly infectable and express relatively low amounts of the receptor on their surfaces (Kadan, et al., J. Virol. 66:2281-2287 (1992).
A cDNA clone (termed W1) encoding a medium ecotropic retroviral receptor (ERR) (SEQ ID NO:4) was identified (Albritton, L. W. et al., Cell 57:659-666 (1989)). The ERR was postulated to be ecotropic murine specific viral receptor for the MuLV retrovirus.
Viral Receptor Mediated Tissue Specificity
Viral receptors can provide tissue specificity for susceptibility to viral infection by tissue specific expression of cell surface proteins that act as specific viral receptors. HIV is an example of a virus exhibiting receptor-mediated tissue restriction, apparently based on its use of the CD4 protein as its primary receptor. Cell receptor concentration is a predominant factor, e.g., the concentration of CD4 receptors on the surface of mouse NIH 3T3 cells is not sufficient to make these cells susceptible to infection by HIV.
The tea (T cell early activation, SEQ ID NO:5) gene, as exemplified by clone 20.5 of MacLeod et al. (J. Biol. Chem. 1:371-279 (1990)), was the first example of a cloned gene or cDNA that has the potential to encode a multiple transmembrane-spanning protein which is induced during T cell activation (Crabtree, Science 243:355-361 (1989)). The function of the tea gene is not yet known.
The sequence of 20.5 cDNA (SEQ ID NO:5) was found to be strikingly homologous to the murine ERR cDNA clone (SEQ ID NO:3) discussed above (Albritton, supra the Rec-1 gene). Retroviral binding and infection studies are required to determine whether the tea-encoded protein functions as a viral receptor (Rein et al., Virology 136:144-152 (1984)). Despite the high degree of similarity between tea and ERR, the two genes differ in chromosomal location, and their predicted protein products differ in tissue expression patterns.
Use of Viruses as Delivery Vectors
The investigation of how viruses replicate and how their genes code for viral proteins has lead to their use as engineered viral vectors for delivering DNA into cells, in vitro, or in situ for the purpose of expressing heterologous DNA in target cells. Thus, viral vectors have been used as delivery vectors to specifically infect and deliver potentially therapeutic or diagnostic DNA or RNA into target cells, based on the target cell specificity of the virus. Viruses have been found to have specific host ranges, termed viral host cell ranges, wherein the trophism of a particular virus is found to be species or tissue specific.
Such viral host cell ranges have been best characterized in the case of the Moloney murine luekemia virus (MLV) which has been most extensively used as a delivery vector for delivery of a heterologous nucleic acid into cells which correspond to the viral host cell range of the MLV. For example, ecotropic MLV will only infect certain types of mouse cells. However, amphotropic MLV as a broad host range, such that amphotropic MLV viruses also infect cells of species other than murine, such as, but not limited to, human.
Accordingly, amphotropic MLV has been most widely used in the laboratory to deliver specific human DNA sequences into target human cells or murine cells. However, to prevent viral replication and further infection in target cells, amphotropic MLV viral vectors have been modified by genetic engineering to be incapable of replicating, by deletion of nucleic acid encoding the env protein. Such engineered amphotrophic MLV have been proposed and preliminarily used to infect human cells in vitro. This MLV is also proposed to be used in vivo human gene therapy, but suffers potential problems of non-specific infection, reversion to replication, competence and/or cancer induction.
Gene Therapy and Gene Transfer
One in every hundred newborn children is born with a serious genetic disorder (Verma, Sci. Amer. 262:68-84 (1990). Often, the effect is accomplished by physical or mental abnormalities, pain and early death (Verma, supra). Because no effective therapies exist for most of the 4,000 known inherited disorders, gene therapists have long sought methods to introduce healthy genes into patients to replace defective genes, or simply to substitute their functions. Advances in recombinant DNA technology, which have made possible the isolation of many genes, as well as much progress in understanding gene regulation, have made this once remote goal possible in the near future. Indeed, over the past several years, the field has seen an enormous amount of progress.
Gene therapy is still in its infancy and many problems remain to be solved. Several areas need further study; such as, but not limited to, gene expression and safety as well as direct, targeted, in viro delivery. The development of vectors that can be safely and efficiently injected directly into patients is a problem that for which there has been a long felt need without suitable solutions. Gene therapy's impact will be limited so long as the technique is carried out as it is currently, where cells are removed from a patient, and the desired gene is transferred in vitro to these cells, which are then returned to the patient. The procedure is also very expensive, and requires too much scientific and medical expertise to be used extensively except in major medical centers (Anderson, W. F. Science 256:808-813 (1992)).
Gene therapy will have a major impact on health care only when vectors are developed that can be safely and efficiently injected directly into patients, a drugs like insulin are now (Anderson, W. F., supra). Vectors need to be discovered and developed that will target specific cell types, insert their genetic information into a safe site in the genome and be regulated by normal physiological signals (Anderson, W. F., supra).
One relatively efficient but problematic means for achieving transfer of genes is by amphotropic retrovirus-mediated gene transfer (see, e.g., Gilboa, E., Bio-Essays 5:252-258 (1987); Williams, D. A. et al., Nature 310:476-480 (1984); Weiss, R. A. et al., RNA Tumor Viruses, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1985). Recombinant amphotropic retroviruses have been used and studied for the possibility of being used as vectors for the transfer of genes into human cells (Cone, R. D. et al., Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984); Danos, O. et al., Proc. Natl. Acad. Sci. USA 6460-6464 (1988)). Such amphotropic viruses, e.g., murine amphotropic MuLV, are capable of infecting human cells. One of the safety problems inherent in this approach which may preclude progress in the clinic, is the fact that even amphotropic retroviruses that have been rendered replication-defective are sometimes capable of generating wild-type variants through recombinational events which provide replication ability (see, e.g., Thompson, Science 257-1854 (1992)). Such an alteration could lead to the widespread retroviral infection in cells and tissues which were not intended to be genetically modified, (Mulligan, Science 257:1854, 1937). Generalized disease could result, such as, but not limited to, cancer or other pathologies caused by insertion of the amphotropic virus' nucleic acid with the LTR's into important functioning genes within a cell which disruption could lead to a pathologic state (Mulligan, R., Science, 260:926 (1993). It is to these needs and problems that the present invention is also directed.
Accordingly, there is a need to overcome one or more problems associated with the use of known retroviruses or other viral vectors for introducing heterologous genes into eukaryotic cells. There is also a need to provide viral receptor proteins which bind viruses and which can be used in diagnostic and/or therapeutic applications without known problems, such as, but not limited to, immunogenicity, blood clearance and non-specific cell binding found with the present use of murine amphotropic viruses.
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